Inappropriate inflammasome activation contributes to multiple human diseases, including autoimmune, autoinflammatory, and metabolic diseases. Yet the mechanisms by which inflammasomes are suppressed are poorly understood. A20 (or TNFAIP3) is a potent anti-inflammatory protein that regulates ubiquitin dependent signals, and is genetically linked to multiple autoimmune and autoinflammatory diseases in humans. Our recent studies show that A20-deficient macrophages exhibit spontaneous NLRP3 inflammasome activity in response to LPS alone without the need for a second signal (e.g., ATP). We have also discovered that IL1? undergoes ubiquitination. Hence, our central hypothesis is that A20 restricts activating ubiquitination events and inflammasome activity in innate immune cells and protects against inflammatory diseases. To test this hypothesis, we will pursue the following specific aims. First, we will identify how A20 regulates a novel TRIF- RIP1-RIP3-Caspase dependent pathway of NLRP3 inflammasome activation. We will determine the in vivo consequences of this A20 function by analyzing the pathophysiology of mice deficient for both A20 and inflammasome components or the downstream pro-inflammatory cytokines. Second, we will utilize a combination of mass spectrometry and novel A20 knockin mutants to precisely define how A20 regulates inflammasome ubiquitination. We will also determine how RIP3 supports IL1? ubiquitination. Finally, we will identify the E3 ubiquitin ligase(s) that ubiquitinate IL-1? and how the A20 binding partner, TXBP, works with A20 to regulate IL1? ubiquitination. Our studies should define new mechanisms by which ubiquitination and A20 regulate inflammasomes, thereby providing a new potential therapeutic pathway targeting inflammasome- mediated diseases.